4-N, 2&#39;-N and 4,2&#39;Di-N-fortimicins AH and AI

ABSTRACT

4-N, 2&#39;-N and 4,2&#39;-di-N-derivatives of fortimicins AH and AI are provided. The compounds are useful as antibacterial agents.

BACKGROUND OF THE INVENTION

The aminoglycoside antibiotics are a valuable therapeutic class of antibiotics which include the kanamycins, gentamicins, streptomycins, sagamicins and the more recently discovered fortimicins. While the naturally produced parent antibiotics are generally, in themselves, valuable antibiotics, chemical modifications have been found to improve the activity, either intrinsic activity or activity against resistant strains or against one or more strains the parent antibiotic is not effective against. Thus, chemical modification has provided both alternative therapeutic agents as well as those which are held in reserve because of the resistance problem. And, because of the development of aminoglycoside-resistant strains and inactivation of the parent antibiotics by R-mediated factors which can develop, the search for new therapeutic entities continues.

Further, some of the naturally produced, parent antibiotics, such as fortimicin B and fortimicin E, are primarily useful as intermediates in preparing derivatives which have more potent antibacterial properties than their weakly active parent antibiotics. The present invention provides derivatives of two such fortimicins fortimicins AH and AI.

Fortimicins AH and AI are two minor factors co-produced in the fermentation of Micromonospora olivoasterospora ATCC No. 21819, 31009 or 31010 according to the method of Nara et al. U.S. Pat. Nos. 3,931,400 and 3,976,768 which disclose the production of fortimicin A and B; along with fortimicin A, fortimicin B and a number of other minor factors which are the subject of copending, commonly assigned patent application Ser. Nos. 025,241; 025,243; 025,247; 025,250; 025,251; and 025,252 filed of even date herewith and with the minor factors disclosed and claimed in commonly assigned, copending U.S. patent application Ser. Nos. 863,015 and 863,016, both filed Dec. 21, 1977.

The 4-N-derivatives of fortimicin B are disclosed in U.S. Pat. No. 4,091,032. The 2'-N and 4,2'-di-N-derivatives of fortimicin B and fortimicin E are disclosed in commonly assigned, co-pending U.S. patent application Ser. Nos. 863,012 and 863,010, both filed Dec. 21, 1977.

SUMMARY OF THE INVENTION

The present invention provides 4-N, 2'-N and 4,2'-di-N fortimicins AH and AI. The compounds of this invention are useful as antibiotics against susceptible gram positive and gram negative bacilli such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillis subtilis, Proteus vulgaris, Shigella sonnei, Salmonella typhi and Klebsiella pneumonia.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compounds of this invention are prepared from fortimicins AH and AI which are represented by Formula I and II: ##STR1##

Fortimicins AH and AI are produced by the fermentation of Micromonospora olivoasterospora as detailed in Examples 1-4.

The compounds of this invention are 4-N-, 2'-N and 4,2'-di-N-fortimicin derivatives and are represented by Formula II: ##STR2## wherein R is hydrogen, acyl, aminoacyl, diaminoacyl, N-loweralkylaminoacyl, N,N-diloweralkylaminoacyl, hydroxy-substituted aminoacyl, loweralkyl, aminoloweralkyl, diaminoloweralkyl, hydroxyloweralkyl, N-loweralkylaminoloweralkyl, aminohydroxyloweralkyl, N,N-diloweralkylaminoloweralkyl, N-loweralkylaminohydroxyloweralkyl, or N,N-diloweralkylaminohydroxyloweralkyl and the pharmaceutically acceptable salts thereof.

The term "acyl", as used in the above definitions refers to acyl radicals of loweralkylcarboxylic acids represented by the formula ##STR3## wherein R is loweralkyl, i.e., acetyl, propionyl, butyryl, valeryl, etc.

The terms aminoacyl, hydroxy-substituted aminoacyl, etc., enumerated in the definitions of R and R₁ for formula II include, but are not limited to as will be obvious to those skilled in the art, naturally occuring amino acids such as glycyl, valyl, alanyl, sarcosyl, leucyl, isoleucyl, prolyl, seryl, and like amino acid residues as well as groups such as 2-hydroxy-4-aminobutyryl and like groups. The amino acid residues included in the above terms, with the exception of glycyl, can be either in the L- or D-configurations or mixtures thereof.

The term "loweralkyl", as used herein, refers to straight or branched chain alkyl radicals containing from 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl and the like radicals.

The term "pharmaceutically acceptable salts refers to the non-toxic acid addition salts of the compounds of Formulae I and II which can be prepared either in situ during the final isolation and purification or by separately reacting the free base with a suitable organic or inorganic acid by methods well known in the art. Representative salts include the mono-, di-, tri-tetra, or other per-salts such as the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate and like salts.

The antibiotics of Formula II are effective antibaterial agents against susceptible or sensitive strains of gram-negative and gram-positive bacilli such as Staphylococcus aureaus, Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Proteus vulgaris, Shigella sonnei, Salmonella typhi and Klebsiella pneumoniae. The compounds of Formula II are administered parenterally, i.e., intravenously, intramuscularly, intraperitoneally or subcutaneously for systemic effect in daily dosages of from 20 to 40 mg/kg of body weight daily, preferrably from 25 to 30 mg/kg of body weight daily based on lean body weight as is good medical practice with the aminoglycoside antibiotics and are preferrably administered in divided dosages. The compounds can also be administered orally at the above dosages to sterilize the intestinal tract and can further be administered in suppository form.

The term "sensitive or susceptible strains" refers to strains of bacilli or organisms which have been demonstrated to be sensitive to a particular antibiotic in a standard in vitro sensitivity test and thus in vitro activity has been established for a particular antibiotic against a specific strain of a specific organism.

Fortimicins AH and AI can be prepared by the fermentation of Micromonospora olivoasterospora ATCC No. 21819, 31009 or 31010 according to the methods described by Nara et al. in U.S. Pat. Nos. 3,931,400 and 3,976,768 for the fermentation of fortimicin A and fortimicin B, and set forth in Examples 1-4 for the fermentation and isolation of fortimicins AH and AI.

The 4-N-acyl fortimicin AI derivatives are prepared following the general procedure used for the preparation of 4-N-acyl derivatives of fortimicins having the fortimicin E stereochemistry for the 4-N-position as disclosed in commonly assigned, co-pending U.S. application Ser. No. 863,010, filed Dec. 21, 1977.

Generally speaking, the 4-N-acyl derivatives can be prepared by reacting 1 mole of salicylaldehyde with fortimicin which results in the formation of 1-N-salicylaldehyde Schiff base fortimicin AI. The latter can then be aminoacylated by coupling the Schiff base intermediate with a variety of activated carboxylic acid derivatives such as a carboxylic acid anhydride, a carboxylic acid chloride, an active carboxylic acid ester or a carboxylic acid azide.

The active esters may be conveniently prepared by reacting the appropriate carboxylic acid, RCOOH with, for example 1-hydroxybenzotriazole, N-hydroxysuccinimide or N-hydroxy-5-norbornene-2,3-dicarboximide according to the method of M. Fugino et al., Chem Pharm Bull, Japan 22: 1857 (1974) wherein R is as defined in formula II for acyl and acyl-containing groups.

For example, the Schiff base fortimicin AI can be aminoacylated with an active ester represented by the formula A--R--Z i.e., N-benzyloxycarbonylglycyl-N-hydroxysuccinimide active ester (A=ONS, R=COCH₂ NH--), N-benzyloxycarbonyl-β-alanyl-N-hydroxy-5-norbornene-2,3-dicarboximide active ester (A=ONB, R=COCH₂ CH₂ NH--), N-benzyloxycarbonylsarcosyl-N-hydroxy-5-norbornene-2,3-dicarboximide active ester (A=ONB), R=COCH₂ N(CH₃₋)-), and N-benzyloxycarbonyl-L-(2-hydroxy-4-amino)butyryl-N-hydroxy-5-norbornene-2,3-dicarboximide active ester (A=ONB, R=COCH(OH)CH₂ CH₂ NH--) where the symbol Z refers to the benzyloxycarbonyl group ##STR4## ONB refers to N-hydroxynorbornyldicarboximide and ONS refers to N-(benzyloxycarbonyloxy)succinimide.

After the above illustrative couplings, the following intermediates are obtained:4-N-(N-benzyloxycarbonyglycyl)-1-N-salicylaldehyde Schiff base fortimicin AI; 4-N-(N-benzyloxycarbonyl-beta-alanyl)-1-N-salicylaldehyde Schiff base fortimicin AI; 4-N-(N-benzyloxycarbonylsarcosyl)-1-N-salicylaldehyde Schiff base fortimicin AI; and 4-N-[N-benzyloxycarbonyl-(L-2-hydroxy-4-aminobutyryl)]-1-N-salicylaldehyde Schiff base fortimicin AI respectively.

It will be readily apparent to those skilled in the art that by substituting the appropriate R group, any of the acyl-containing intermediates for the corresponding final products can be obtained.

The Schiff base intermediates are treated with 0.2 N aqueous hydrochloric acid to cleave the Schiff base protecting groups and the resulting crude hydrochloride salts are subjected to silica gel chromatography in a solvent system containing ammonium hydroxide which results in the following illustrative, partially deprotected intermediates:4-N-(N-benzyloxycarbonylglycyl)fortimicin AI; 4-N-(N-benzyloxycarbonyl-beta-alanyl)-fortimicin AI; 4-N-(N-benzyloxycarbonylsarcosyl)fortimicin AI; and 4-N-[N-benzyloxycarbonyl-(L-2-hydroxy-4-aminobutyryl)]-fortimicin AI. The 4-N-protected intermediates are then reacted with N-benzyloxycarbonyloxy-5-norbornene-2,3-dicarboximide (Z-ONB) to form the corresponding di-N-protected intermediates, i.e. di-N-benzyloxycarbonyl-4-N-glycylfortimicin AI.

Hydrogenolysis of the di-N-protected intermediates over palladium on carbon catalyst (5% Pd/C) in, for example 0.2 N hydrochloric acid in methanol yields the desired final products, i.e. 4-N-glycylfortimicin AI dihydrochloride, 4-N-sarcosylfortimicin AI dihydrochloride, etc.

4-N-alkylation is readily accomplished by reducing the corresponding acyl, hydroxyacyl or amino-containing acyl product with diborane.

4-N-acylation of fortimicin AH is accomplished by the method disclosed in U.S. Pat. No. 4,091,032 which discloses 4-N-fortimicin B derivatives.

It is understood that the terms acyl and alkyl have, for the purpose of the above discussion have been used as shorthand references to the terms "loweralkyl" and "acyl" defined in the specification and to the acyl and alkyl-containing definitions for R and R₁ is Formula II. This shorthand reference has been used to simplify the above discussion, not to modify the terms as defined.

The following Examples further illustrate the present invention by setting forth the fermentation and isolation of fortimicins AH and AI which are coproduced with fortimicin A, fortimicin B, isofortimicin, fortimicin E and a number of other minor factors.

Fortimicins AH and AI can be prepared by the fermentation of Micromonospora olivoasterospora ATCC 21819 in a suitable fermentation broth and isolated as described hereinbelow.

EXAMPLE 1 Preparation of Fermentation Broth

6000 Liters of a fermentation broth having the following composition and pH 7 before sterilization is prepared:

    ______________________________________                                         Ingredient       Weight Percent                                                ______________________________________                                         Starch          4.00                                                           Soybean meal    2.00                                                           Cornsteep liquor                                                                               0.05                                                           K.sub.2 HPO.sub.4                                                                              0.05                                                           MgSO.sub.4 . 7 H.sub.2 O                                                                       0.05                                                           KC1             0.03                                                           CaCO.sub.3      0.1                                                            Water           to 100.00                                                      ______________________________________                                    

EXAMPLE 2 Preparation of Inoculum

Micromonospora olivoasterospora ATCC 21819 is used as a seed strain and is initially cultured in a first seed medium containing 2% glucose, 0.5% peptone, 0.5% yeast extract and 0.1% calcium carbonate (pH 7.2 before sterilization) by inoculating one loopful of the seed strain into 10 ml of the seed medium in a 50 ml large test tube. Culturing is carried out at 30° C. for 5 days with shaking. Ten ml of the seed culture broth is then inoculated into 30 ml of a second seed medium in a 250 ml Erlenmeyer flask. The composition of the second seed medium is the same as that of the first seed medium. The second seed culturing is carried out at 30° C. for two days with shaking.

Then 30 ml of the second seed culture broth is inoculated into 300 ml of a third seed medium in a two liter Erlenmeyer flask provided with baffles. The composition of the third seed medium is the same as that of the first seed medium and the third seed culturing is carried out at 30° C. for 2 days with shaking. Thereafter, 1.5 liters of the third seed culture broth (corresponding to the contents of five flasks) is inoculated into 5 liters of a fourth seed medium in a 30 liter glass jar fermenter. The composition of the fourth seed medium is the same as that of the first seed medium. Culturing in the jar fermenter is carried out at 30° C. for two days with aeration (15 liters/min) and stirring (350 r.p.m.).

EXAMPLE 3 Production of Fortimicins AH and AI

Fifteen liters of the fourth seed culture broth of Example 2 is inoculated into 150 liters of a main fermentation medium in a 300 liter stainless steel fermenter. The main fermentation medium comprises: 4% starch, 2% soybean meal, 1% corn steep liquor, 0.05% K₂ HPO₄, 0.05% MgSO₄.7H₂ O, 0.3% KCl and 0.1% CaCO₃ and water. (pH 7.0 before sterilization) Culturing in the fermenter is carried out at 30° C. for 4 days with aeration (80 liters/min) and stirring (150 r.p.m.).

EXAMPLE 4 Isolation of Fortimicins AH and AI

To 5000 liters of the fermentation broth, prepared as described above, is added 102 liters of a weakly acidic carboxylic (polymethacrylate) type cation exchange resin in the ammonia form, e.g. Amberlite IRC-50 sold by the Rohm and Haas Company. The mixture is agitated for two hours, during which time the mixture is maintained at pH 6.6 by the addition of sulfuric acid. The ion exchange resin is separated from the broth by centrifugation and then added to a column and backwashed with deionized water until free of extraneous solids. The column is washed with water, then eluted downflow with 1 N ammonium hydroxide. Elutes of pH 9.6 to about 11.3 are collected and concentrated under reduced pressure until excess ammonia is removed. The solution is adjusted to pH 2.0 with hydrochloric acid and treated with 5%(w/v) activated carbon such as Pittsburg RB carbon sold by Calgon Corporation. The solution is then filtered through a diatomaceous earth mat and the filtrant concentrated under reduced pressure to give a mixture of crude fortimicins in methanol (5 liters).

A portion of the crude fortimicins in methanol (5 liters) is chromatographed on a column of silica gel (4 kg) eluted with methanol with an increasing gradient concentration of hydrochloric acid. Initial fractions are combined, adjusted to pH 5 and concentrated to yield 237 g (dry weight) of crude fortimicin components. A portion (40 g of the crude component mixture is dissolved in water and applied to a Bio Rad AG 2X8 resin(OH⁻) form column and eluted with water. The fractions giving positive ninhydrin spots on thin layer chromatography (tlc) plates are combined and concentrated to give 17 g of solids. These are dissolved in water and chromatographed on a column (3.5 cm diam. x 47 cm) of Bio Rex 70 resin (NH₄ ⁺ form) developed with a gradient from water (one liter) to 1 N ammonium hydroxide (1 liter). Initial fractions are combined and concentrated to yield 5.4 g of solids which are rechromatographed in two portions over a similar column of Bio RNx 70 resin (NH₄ ⁺ form). Fractions containing the desired components are pooled to give two concentrates containing fortimicin AH (earlier fractions) and AI (later fractions).

EXAMPLE 5 Purification of Fortimicin AH

Crude fortimicin AH(260 mg) is purified by chromatography on a column (1.5 cm diameter×61 cm) of E. Merck silica gel 60 developed with the lower phase of a mixture of chloroform, methanol, ammonium hydroxide[1.5:1:1(v/v/v)]. Fractions containing pure fortimicin AH as detected on tlc plates are combined and concentrated to yield pure fortimicin AH(46.8 mg). PMR spectrum in deuterium oxide with external tetramethylsilane as reference: δ2.65 (3H) singlet CH₃ -C_(7') ; δ 2.84 (3H) singlet NCH₃ ;δ 3.90 (3H) singlet OCH₃ ;δ 5.71 (1 H) doublet C_(1') H.

EXAMPLE 6 Purification of Fortimicin AI

Crude fortimicin AI (430 mg) is chromatographed on a column of E. Merck silica gel in the lower phase of a mixture of chloroform-methanol-ammonium hydroxide[1.5:1:1 (v/v/v)] and fractions containing fortimicin AI are combined and concentrated to yield 265 mg of solid which is rechromatographed on a column of silica gel developed with a mixture of chloroform-methanol-ammonium hydroxide, initially 90:10: 4(v/v/v) followed stepwise by 70:30:0.4(v/v/v) and 70:30:0.8 (v/v/v). Fractions containing fortimicin AI are combined and concentrated to yield 60 mg which is prurified by further chromatography over silica gel in chloroform-methanol-ammonium hydroxide[70:30:1.6(v/v/v)] to give fortimicin AI(43.3 mg). Proteon magnetic resonance spectrum in deuterium oxide with tetramethylsilane as external reference:δ 2.65 (3H) singlet C_(7') CH₃, δ 2.89 (3H) singlet NCH₃, δ 4.03 (3H) singlet OCH₃ ; δ 5.75 (1H) doublet C_(1') H.

The following examples illustrate the present invention.

EXAMPLE 7 1,2'-Di-N-salicylaldehyde Schiff base fortimicin AI

A solution of 1.3 g of fortimicin AI and 1.03 g of salicylaldehyde in 30 ml of methanol is refluxed and stirred for 1 hour. The solvent is evaporated under reduced pressure and the residue is dissolved in 30 ml of benzene which is likewise evaporated under reduced pressure. This last process is repeated six times. The residue is dried under high vacuum over KOH pellets to yield the desired product.

EXAMPLE 8 4-N-(N-Benzyloxycarbonylglycyl)-1,2'-di-N-salicylaldehyde Schiff base fortimicin

A solution of the above prepared 1,2'-di-N-salicylaldehyde Schiff base fortimicin AI (2.8 g) and N-benzyloxycarbonylglycyl-N-hydroxysuccinimide active ester (2 g) in 25 ml of tetrahydrofuran is stirred at room temperature overnight. The solvent is evaporated under reduced pressure to afford a residue of about 4 g of product.

EXAMPLE 9 4-N-(N-Benzyloxycarbonylglycyl)fortimicin AI

The substance obtained in Example 6 (4 g) is dissolved in 500 ml of chloroform and the solution is shaken with 500 ml of 0.2 N aqueous hydrochloric acid. The layers are separated and the chloroform solution is extracted with three 150 ml portions of 0.2 N aqueous hydrochloric acid. The hydrochloric acid extracts are washed in series with three 250 ml portions of chloroform. The chloroform solutions are dried over anhydrous sodium sulfate, filtered, combined and evaporated to leave a residue of nonbasic material which is not characterized.

The aqueous hydrochloric acid extracts are evaporated under reduced pressure at room temperature. The residue is dissolved in 60 ml of methanol and the solvent is likewise evaporated. This last process is repeated six times. The residue is dried over potassium hydroxide pellets under high vacuum to afford crude 4-N-(N-benzyloxycarbonylglycyl) fortimicin AI dihydrochloride salt.

A partial purification of the above residue (2.0 g) by chromatography on 270 g of silica gel using the lower phase of a mixture of chloroform-methanol-concentrated aqueous ammonium hydroxide [1:1:1(v/v/v)] as the eluting solvent system affords a mixture containing the desired product. Further chromatography of this residue (ca 1.4 g) on 180 g of silica gel using the lower phase of a chloroform-methanol-concentrated aqueous ammonium hydroxide-water mixture[2:2:1:1(v/v/v/v)] as the solvent system leads to the separation of several components. Evaporation of the solvent from the early chromatographic fractions leads to the isolation of nonpolar substances which are not further characterized. A next group of fractions yields a small quantity of 4,2'-di-N-(N-benzyloxycarbonylglycyl)fortimicin. Later fractions of the chromatogram result in the desired product.

EXAMPLE 10 N-Oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide

To an ice cooled suspension of 30 g of N-hydroxy-5-norbornene-2,3-dicarboximide in 150 ml of water are added 7.06 g of sodium hydroxide pellets over a period of 10 minutes with stirring. Methanol is added to the ice bath to bring the temperature to -5° C. and the contents of the flask are stirred for 10 minutes. Twenty-three ml of benzyloxycarbonyl chloride are then added to the stirred solution over a period of 15 minutes. The mixture is then stirred at -5° C. for 2 hours and then at room temperature for 24 hours. The reaction mixture is extracted with 400 ml of chloroform and the chloroform extract washed with three 200 ml portions of water. The aqueous washes are then extracted in series with four 200 ml portions of chloroform. The chloroform extracts are dried over anhydrous magnesium sulfate, filtered, combined and evaporated to leave a residue of 39.88 g. The crude material is recrystallized from 95% ethanol. The crystals which form upon cooling are collected on a filter and washed with several small portions of cold ethanol. After drying, 29.11 g of crystalline product is obtained, m.p.126°-127° C. A sample is recrystallized twice more for analysis:m.p. 126°-127° C.; IR(CDCl₃) 1800 (shoulder), 1782, 1732 cm⁻¹ : PMR (CDCl₃) 7.41(Z-Ar), 6.2 (vinyl H), 5.31 (CH₂ -Z), 3.4 (H single proton), 1.7 (CH₂)ppm.

Anal. Calcd. for C₁₇ H₁₅ NO₅ : C,65.17; H,4.83; N, 4.47. Found: C,65.02; H,4.82; N, 4.26.

EXAMPLE 11 1,2',4"-Tri-N-benzyloxycarbonyl-4-N-glycylfortimicin AI

A solution containing 1.03 g of the above prepared 4-N-(N-benzyloxycarbonylglycyl)fortimicin AI and 2 g of N-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarbonximide in 56 ml of methanol are stirred at room temperature overnight. Evaporation of the solvent under reduced pressure leaves a residue of about 3 g of crude reactoin mixture. The latter is purified by repeated silica gel column chromatography using benzene-methanol-ethanol [1170:34:136(v/v/v)] and benzene-chloroform-ethyl acetate-n-propanol[13:16:8:3(v/v/v/v)] as the eluting systems.

Combination of the appropriate fractions following the minor unidentified somponenet in the original chromatograms and evaporation of the solvents leave a residue of the desired product. An analytical sample can be obtained by rechromatography of a part of the above product on silica gel using benzene-methanol-ethanol[1170:36:136(v/v/v)] as the eluant.

EXAMPLE 12 4-N-Glycylfortimicin AI trihydrochloride salt

A solution of 0.5 g of tri-N-benzyloxycarbonyl-4-N-glycylfortimicin AI in 34 ml of 0.2 N hydrochloric acid in methanol and 16 ml of methanol is hydrogenolyzed over 5% Pd/C for 4 hours. The catalyst is collected on a filter ans washed with methanol. The filtrate is evaporated under reduced pressure, the residue redissolved in methanol and this solvent evaporated. This last procedure is repeated six times to yield the desired product after drying in vacuo over potassium hydroxide pellets.

EXAMPLE 13 4-N-(N-Benzyloxycarbonyl-β-alanyl) fortimicin AI

A solution of 2.579 g of the compound of Example 5 and 2.5 g of the N-benzyloxycarbonyl-β-alanyl ester of N-hydroxy-5-norbornene-2,3-dicarboximide, prepared by the method of M. Fujino et al., Chem Pharm. Bull. Japan, 22, 1857(1974), in 30 ml of tetrahydrofuran is stirred at room temperature for 25 hours. Evaporation of the solvent under reduced pressure yields about 5 g of crude product.

This material is dissolved in 500 ml of chloroform and the solution is shaken with 500 ml of 0.2 N hydrochloric acid. The layers are separated and the chloroform solution is extracted with three 150 ml portions of 0.2 N hydrochloric acid. The acid extracts are washed in series with three 25 ml portions of chloroform. The chloroform washes are dried over anhydrous magnesium sulfate, filtered, combined and evaporated to yield a non-basic material. The acidic extracts are combined and evaporated under reduced pressure at low temperature. The residue is dissolved in 30 ml of methanol which is also evaporated. This last process is repeated six times to yield a residue of reaction products as the hydrochloride salts after drying in vacuo over potassium hydroxide. The above hydrochloride salts are chromatographed on 170 g of silica gel using the lower layer of a chloroform-methanol-concentrated ammonium hydroxide mixture [1:1:1(v/v/v)] as the eluting solvent. Combination of the fractions containing the desired product and evaporation of the solvents affords a residue which is rechromatographed on 170 g of silica gel using the lower phase of a chloroform-methanol-concentrated ammonium hydroxide-water [2:2:1:1(v/v/v/v)] mixture as the eluting solvent. After combination and evaporation of the solvents from the appropriate fractions, the desired product is obtained.

EXAMPLE 14 Tri-N-benzyloxycarbonyl-4-N-β-alanylfortimicin AI

A solution of O 75 g of the above prepared 4-N-(N-benzyloxycarbonyl-β-alanyl)fortimicin AI and 1.440 g of N-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide in 40 ml of methanol is stirred at room temperature for 24 hours. Evaporation of the solvent leaves a residue which is chromatographed on 180 ml of silica gel using a methylene chloride-methanol-concentrated ammonium hydroxide mixture [185:15:2(v/v/v)] as the eluent. Combination of the fractions containing the desired substance and evaporation of the solvent leaves a partially purified product. Repeated chromatography of this sample on silica gel using benzene-methanol-ethanol[1170:34:136(v/v/v)], methylene chloride-methanol-concentrated ammonium hydroxide[185:15:2(v/v/v)] yields the desired product.

EXAMPLE 15 4-N-β-Alanylfortimicin AI trihydrochloride AI

A solution of 0.35 g of the compound of Example 12 in 30 ml of 0.2 N hydrochloric acid in methanol and 20 ml of methanol is hydrogenolyzed over 0.36 g of 5% Pd/C for 4 hours. The catalyst is collected on a filter and washed with several small portions of methanol. The filtrate is evaporated under reduced pressure and the residue is dissolved in 30 ml of methanol which is likewise evaporated under reduced pressure. This last process is repeated six times to afford a residue of about 0.2 g of the desired product after drying in vacuo over potassium hydroxide pellets.

EXAMPLE 16 N-Benzyloxycarbonylsarcosyl active ester of N-hydroxy-5-norbornene-2,3-dicarboximide

To a solution of 4.471 g of N-benzyloxycarbonylsarcosine and 3.754 g of N-hydroxy-5-norbornene-2,3-dicarboximide in 15 ml of tetrahydrofuran and 15 ml of dioxane is added N,N-dicyclohexylcarbodiimide in 2 ml of tetrahydrofuran and 2 ml of dioxane according to the method of M. Fujino et al., Chem Pharm. Bull., Japan, 22, 1857(1974). The mixture is stirred at room temperature overnight. The dicyclohexylurea which precipitates from the reaction mixture is collected on a filter and washed with a total of 20 ml of tetrahydrofuran-dioxane[1:1(v/v)]. Evaporation of the solvent from the filtrate affords 8.523 g of crude product. The substance is recrystallized from isopropanol to yield 4.294 g of the active ester, m.p. 75°-80° C. Concentration of the mother liquors yields an additional 4.125 g of less pure product, m.p. 69°-73° C.

A portion of the first crop is recrystallized for analysis: m.p. 80°-82° C.; IR(CDCl₃) 1821,1774,1725,1700(shoulder) cm⁻¹ ; NMR (CDCl₃) 7.32(Ar-Z), 6.17(vinyl), 5.13(CH₂ -Z), 4.3(sar-CH₂), 3.35(single H), 3.0(sar-CH₃),1.64(--CH₂) ppm. Anal. Calcd. for C₂₀ H₂₀ N₂ O₆ : C, 62.49; H, 5.24; N, 7.29. Found: C, 62.65; H, 5.28; N, 7.24.

EXAMPLE 17 4-N-(N-Benzyloxycarbonylsarcosyl)fortimicin AI

A solution of 2.4 g of the compound of Example 5 and 2.5 g of the N-benzyloxycarbonylsarcosyl active ester of N-hydroxy-5-norbornene-2,3-dicarboximide of Example 14 in 25 ml of tetrahydrofuran is stirred for 24 hours at room temperature. Evaporation of the solvent under reduced pressure affords a residue of crude product. The residue is taken up in 500 ml of chloroform and the solution is shaken with 500 ml of 0.2 N aqueous hydrochloric acid. The layers are separated and the chloroform solution is extracted with three 150 ml portions of 0.2 N hydrochloric acid. The acid extracts are washed in series with three 250 ml portions of chloroform. The chloroform washes are dried over anhydrous sodium sulfate, filtered, combined and evaporated to leave a non-basic residue.

The acidic extracts are combined and evaporated under reduced pressure at low temperature. The residue is taken up in 30 ml of methanol which is likewise evaporated and the procedure repeated six times. The resulting residue is dried in vacuo over potassium hydroxide pellets.

The above residue is chromatographed on 160 g of silica gel using the lower phase of a chloroform-methanol-concentrated ammonium hydroxide mixture [1:1:1(v/v/v)] as the eluting solvent. The fractions containing the desired product are combined and the solvents evaporated leaving crude product which is rechromatographeon 160 g of silica gel using the lower phase of a chloroform-methanol-concentrated ammonium hydroxide-water mixture [2:2:1:1(v/v/v/v)] as the eluting solvent. After combination of the appropriate fractions and evaporation of the solvents the desired product is obtained.

EXAMPLE 18 Tri-N-benzyloxcarbonyl-4-N-sarcosylfortimicin AI

A solution of 0.15 g of the product of Example 15 and 0.3 g of N-oxybenzyloxycarbonyl-5-norbornene 2,3-dicarboximide in 10 ml of methanol is stirred at room temperature overnight. Evaporation of the solvent under reduced pressure affords a residue which is chromatographed on 70 g of silica gel using a benzene-methanol-ethanol mixture [1170:34:136(v/v/v)] as the eluent. Combination of the appropriate fractions and evaporation of the solvent yields partially purified tri-N-benzyloxycarbonyl-4-N-sarcosylfortimicin AI. A second chromatogram of this substance on 50 g of silica gel, employing the same solvent system yields the desired product in pure form.

EXAMPLE 19 4-N-Sarcosylfortimicin AI trihydrochloride

A solution of 0.15 g of the compound of Example 16 in 12 ml of 0.2 N hydrochloric acid in methanol and 23 ml of methanol is hydrogenolyzed over 0.150 g of a 5% Pd/C catalyst for 4 hours. The catalyst is collected on a filter and washed with methanol. The filtrate is evaporated to dryness under reduced pressure and the residue is dissolved in 20 ml of methanol which is likewise evaporated. The last procedure is repeated six times. The residue is dried in vacuo over potassium hydroxide pellets to afford the desired product.

EXAMPLE 20 4-N-[N-Benzyloxycarbonyl-(L-2-hydroxy-4-aminobutyryl)]fortimicin AI

The N-hydroxy-5-norbornene-2,3-dicaboximide active ester of L-N-benzyloxycarbonyl-2-hydroxy-4-aminobutyric acid is prepared according to the method of M. Fujino et al. supra. To an ice cold solution of 1.645 g of L-N-benzyloxycarbonyl-2-hydroxy-4-aminobutyric acid and 1.182 g of N-hydroxy-5-norbornene-2,3-dicarboximide in 16 ml of tetrahydrofuran-dioxane [1:1(v/v)], are added, with stirring, 1.374 g of N,N-dicyclohexylcarbodiimide and 5 ml of tetrahydrofuran-dioxane [1:1(v/v)]. The mixture is stirred at 0° C. for 50 minutes and then at room temperature for 3 hours.

The N,N-dicyclohexylurea produced by the above reaction is collected on a filter and washed with 10 ml of tetrahydrofuran-dioxane [1:1(v/v)]. The filtrate is collected in a flask containing 2.0 g of the compound of Example 5. The reaction mixture is stirred at room temperature for 20 hours. Evaporation of the solvent leaves crude product.

Five and one-half grams of the crude product is dissolved in 500 ml of chloroform and the solution is shaken with 500 ml of 0.2 N hydrochloric acid. The chloroform phase is separated and extracted with three 150 ml portions of 0.2 N hydrochloric acid. The aqueous phases are washed in series with three 250 ml portions of chloroform. The chloroform solutions are dried over anhydrous sodium sulfate, filtered, combined, and evaporated to afford a residue of non-basic substances.

The aqueous extracts are combined and evaporated in vacuo at low temperature. The residue is dissolved in 30 ml of methanol and the solvent evaporated. This last process is repeated six times and the resulting material is dried over potassium hydroxide pellets in vacuo to yield a residue of crude product.

3.5 g of the crude product are chromatographed on 270 g of silica gel using the lower phase of a chloroform-methanol-concentrated ammonium hydroxide [1:1:1(v/v/v)] mixture as the eluent. The fractions containing the desired substance are combined and evaporation of the solvent yields a residue of partially purified product which is rechromatographed on 180 g of silica gel using the lower phase of a chloroform-methanol-concentrated ammonium hydroxide-water [2:2:1:1(v/v/v/v)] mixture as the eluent. Combination and evaporation of the active fractions affords the desired product.

EXAMPLE 21 Tri-N-benzyloxycarbonyl-4-N-(L-2-hydroxy-4-aminobutyryl)]fortimicin

A solution of 0.8 g of the above prepared substance and 1.5 g of N-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide in 40 ml of methanol is stirred at room temperature overnight. Evaporation of the solvent from the reaction mixture under reduced pressure affords a residue of about 2 g of crude product which is chromatographed on 180 g of silica gel using a mixture of methylene chloride-methanol-concentrated ammonium hydroxide [185:15:2(v/v/v)] as the eluent. The early fractions of the chromatogram contained the desired substance contaminated with less polar components. From the subsequent fractions, the desired product is isolated after evaporation of the solvent. Repeated silica gel chromatography of the residue of the early fractions above using benzene-methanol-ethanol-acetic acid [1170:35:135:10(v/v/v/v)] and ethyl acetate-ethanol [98:2(v/v)] mixtures affords additional product. After two more chromatograms on silica gel using ethyl acetate-ethanol [98:2(v/v)] as the eluent, the desired product is obtained in pure form.

EXAMPLE 22 4-N-(L-2-Hydroxy-4-aminobutyryl)fortimicin AI trihydrochloride

A solution of about 0.2 g of the above prepared compound in 16.3 ml of 0.2 N hydrochloric acid and 8.7 ml of methanol is hydrogenolyzed over a 0.2 g of 5% Pd/C for 4 hours. The catalyst is collected on a filter and washed with methanol. The filtrate is evaporated under reduced pressure and the residue is dissolved in 10 ml of methanol which is likewise evaporated. This last process was repeated six times and the resulting material is dried under high vacuum over potassium hydroxide pellets to afford the desired material.

EXAMPLE 23 Tri-N-benzyloxycarbonyl-4,2'-N,N'-diglycylfortimicin AI

A solution of the substance contained in the chromatographic fractions preceeding the final product of Example 7 and 0.128 g of N-oxybenzyloxycarbonyl-5-norbornene-2,3-dicarboximide in 3 ml of methanol is stirred at room temperature overnight. Evaporation of the solvent affords a residue which is chromatographed in silica gel using benzene-ethanol-isopropanol [9:1:1(v/v/v)] as the eluting solvent. Combination of the appropriate fraction and evaporation of the solvent leaves a residue which is rechromatographed on silica gel using ethyl acetate-methanol [98:2(v/v)] as the eluting solvent to afford the desired product.

EXAMPLE 24 4,2'-N,N'-Diglycylfortimicin AI trihydrochloride

A solution of 0.75 g of the above prepared compound in 6 ml of 0.02 N hydrochloric acid in methanol is hydrogenolyzed over 0.08 g of 5% Pd/C for four hours. The catalyst is collected on a filter and washed with several portions of methanol. The filtrate is evaporated under reduced pressure and the residue redissolved in 15 ml of methanol which is likewise evaporated. This last procedure is repeated six times and the desired product dried in vacuo over potassium hydroxide pellets.

EXAMPLE 25 4-N-(β-Aminoethyl)fortimicin AI

A stirring solution of 4N-glycylfortimicin AI (2.0 g) in tetrahydrofuran (80 ml) is treated with 1.22 g of lithium aluminum hydride. The stirring reaction mixture is refluxed for 20 hours and then the excess lithium aluminum hydride is consumed by the careful addition of water. The insoluble material is sedimented by centrifugation. The pellet is suspended in 50 ml of water and centrifuged. The combined supernatants are taken to dryness under reduced pressure to yield crude product which is chromatographed on a column (2.0×40 cm) of cation exchange resin, carboxylic type, e.g., Bio-Rad Laboratories, Bio-Rex 70,100-200 mesh, ammonia form, and eluted with a gradient of water to 1 N ammonium hydroxide. Fractions containing the desired product are concentrated to a small volume and lyophilized to give the desired product.

EXAMPLE 26 1,6'-Di-N-benzyloxycarbonylfortimicin AH

To a solution of 2.0 g of fortimicin AH free base in 30 ml of water and 50 ml of methanol, cooled in an ice bath and constantly stirred, is added N-(benzyloxycarbonyloxy)succinimide. The reaction is stirred at ice bath temperature for 4 hours and then at room temperature for 22 hours. The reaction is concentrated under reduced pressure and poured into 400 ml of water to which is added 200 ml of chloroform. The organic layer is separated on silica gel prepared and eluted with a solvent system consisting of chloroform-methanol-concentrated ammonium hydroxide [23.4:1.4:0.1(v/v/v)]. Fractions containing pure 1,6'-di-N-benzyloxycarbonylfortimicin AH are collected and evaporated to dryness.

EXAMPLE 27 Tri-N-benzyloxycarbonyl-4-N-glycylfortimicin AH

To a stirred solution of 1.0 g of the above-prepared 1,6'di-N-benzyloxycarbonylfortimicin AH in 6.5 ml of tetrahydrofuran is added 2 moles of N-hydroxysuccinimidyl-N-benzyloxycarbonylglycine. Stirring is continued for 22 hours at room temperature. The reaction is concentrated to dryness under reduced pressure and the resulting product chromatographed on a column of silica gel with a solvent system consisting of benzene-methanol-95% ethanol-concentrated ammonium hydroxide [23.5:1.4:2.0:0.2(v/v/v/v)]. Fractions containing the desired product are taken to dryness to yield the desired product.

EXAMPLE 28 4-N-Glycylfortimicin AH trihydrochloride

Tri-N-benzyloxycarbonyl-4-N-glycylfortimicin AH (0.5 g) in 30 ml of 0.2 N hydrochloric acid in methanol is hydrogenolyzed for 4 hours under 3 atmospheres of hydrogen in the presence of 0.1 g of 5% palladium on carbon. The catalyst is removed by filtration and the filtrate concentrated to dryness under reduced pressure. Excess acid is removed by co-distillation with methanol under reduced pressure to afford the desired product.

EXAMPLE 29 Tri-N-benzyloxycarbonyl-4-N-(β-aminoethyl)fortimicin AH

To an ice cold stirring solution of tri-N-benzyloxycarbonyl-4-N-glycylfortimicin AH is added 1.0 ml of a 1 M solution of diborane in tetrahydrofuran. The reaction mixture is stirred for 3 hours under a nitrogen atmosphere and then treated with an additional 1.0 ml of the diborane solution. After stirring for an additional 2 hours under nitrogen, water is added and the solvents evaporated under reduced pressure. Purification by column chromatography on silica gel prepared and eluted with a solvent system consisting of chloroform-methanol-concentrated ammonium hydroxide [23.4:1.4:0.1(v/v/v)] gives the pure product.

EXAMPLE 30 4-N-(β-Aminoethyl)fortimicin AH trihydrochloride

Tri-N-benzyloxycarbonyl-4-N-(β-aminoethyl)fortimicin AH in 25 ml of 0.2 N hydrochloric acid in methanol is hydrogenolyzed for 4 hours under 3 atmospheres of hydrogen in the presence of 0.15 g of 5% palladium on carbon. The catalyst is removed by filtration and the filtrate concentrated to dryness under reduced pressure. Excess acid is removed by co-evaporation with methanol under reduced pressure to give the desired product.

The in vitro antibiotic activity is determined by a two-fold agar dilution method using 10 ml per petri plate of Mueller-Hinton agar. The agar is inoculated with one loopful (0.001 ml loop) of a 1:10 dilution of a 24 have broth culture of the indicated test organism and incubated at 37° C. for 24 hours.

The compounds of this invention are active as systemic antibiotics when injected by parenteral routes of administration, i.e., by the intramuscular, intravenous, intraperitoneal or subcutaneous routes of administration. The compounds can also be administered orally in those instances where it is desirable to sterilize the intestinal tract and can also be applied topically or in suppository form.

Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying and dispersing agents. They may be sterilized, by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions and the like. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

Solid dosage forms for oral administration including capsules tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Besides, such inert diluents compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient shall be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. Generally, dosage levels of between 20 to 40 mg/kg of body weight daily, based on lean body weight are administered to a mammalian patient suffering from an infection caused by susceptible organism. 

The invention claimed is:
 1. 4-N,2'-N and 4,2'-Di-N-derivatives of fortimicins AI and AH represented by the formulae I and II respectively ##STR5## wherein R is selected from the group consisting of acyl, aminoacyl, diaminoacyl, N-loweralkylaminoacyl, N,N-diloweralkylaminoloweralkyl, hydroxy-substituted aminoacyl, loweralkyl, hydroxyloweralkyl, aminoloweralkyl, diaminoloweralkyl, N-loweralkylaminoloweralkyl, N,N-diloweralkylaminoloweralkyl, N-loweralkylaminohydroxyloweralkyl and N,N-diloweralkylaminohydroxyloweralkyl; and the pharmaceutically acceptable salts thereof.
 2. A compound of claim 1 represented by formula I.
 3. A compound of claim 2: 4-N-glycylfortimicin AI or a pharmaceutically acceptable salt thereof.
 4. A compound of claim 2: 4-sarcosylfortimicin AI or a pharmaceutically acceptable salt thereof.
 5. A compound of claim 2: 4-N-β-alanylfortimicin AI or a pharmaceutically acceptable salt thereof.
 6. A compound of claim 2: 4-N-(L-2-hydroxy-4-aminobutyryl)fortimicin AI or a pharmaceutically acceptable salt thereof.
 7. A compound of claim 2: 4-N-(β-aminoethyl)fortimicin AI or a pharmaceutically acceptable salt thereof.
 8. A compound of claim 1 represented by formula II.
 9. A compound of claim 8: 4-N-glycylfortimicin AH or a pharmaceutically acceptable salt thereof.
 10. A compound of claim 8: 4-N-sarcosylfortimicin AH or a pharmaceutically acceptable salt thereof.
 11. A compound of claim 8: 4-N-β-alanylfortimicin AH or a pharmaceutically acceptable salt thereof.
 12. A compound of claim 8: 4-N-(L-2-hydroxy-4-aminobutyryl)fortimicin AH or a pharmaceutically acceptable salt thereof.
 13. A compound of claim 8: 4-N-(β-aminoethyl)fortimicin AH or a pharmaceutically acceptable salt thereof.
 14. A pharmaceutical composition comprising an anti-bacterially effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier or diluent. 